Bacterial Protein E.P.C (Expression, Purification and Characterization)
Title: GBR22 Protein experiments of expression, purification, and characterization.
Introduction: The use of recombinant proteins in research has increased as the development of new technologies and analysis of protein structure and function used for amplification and purification. Protein expression and purification are key processes in study of genomic information, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the high-throughput screening technologies commonly used in pharmaceutical development [1]. E.Coli bacterial protein would overexpressed and purified for affinity, ion-exchange crystallography, enzyme inhibition assays and other techniques [2]. Concentration was calculated using spectrophotometer and Beer’s law. Three protein labs were to overexpress, purify, and characterize the recombinant pGEM-gbr22 protein.
Materials & Methods: As safety precautions, goggles, gloves, and lab coats were worn even before the pre-lab calculation and food and drinks were prohibited in lab area. The materials used were bacteria E. Coli BL21 (DE3) strain, pGEM-gbr22 protein from NEB, beakers, several test tubes, petri dishes, centrifuge tubes, pipettors and tips and, Erlenmeyer flasks, spectrophotometer (Thermo Scientific, Wilmington, DE), gel electrophoresis device (mini-PROTEAN), centrifuge machine, a and other lab equipments. Procedure was divided into three parts: bacterial protein expression, purification, and characterization. E. Coli bacteria was prepared and 25ul of bacteria was added into 2 transformation tubes: one control and other with DNA. Petri dishes were used and 200ul of SOC media were added after few ice and heat shock process. 50ul of mixture were added to 2 petri dishes and labeled: one control and other with DNA. Plates were stored in 37 ̊C incubator overnight. Bacterial colonies were added to LB broth and left in incubator for 7 hours. Ampicillin was added to the culture in Erlenmeyer flask and left in incubator for a day to grow. 500ul of purple sample 1 was collected from the culture and centrifuged for 5 minutes at 5000 rpm at 4 ̊C. 2.5ml of 1x PBS solution was added to the tube and lysozyme was also added to make the final concentration of 1mg/ml. Wet pellet was weighted 12 0.18g after centrifuge. Sample2 was obtained after the centrifuge of 2ul of benzonase added to solution. The remained liquid was syringe filtered. Wash and elution buffers were made from 1 PBS and 20mM imidazole and 1x PBS and 250mM imidazole. Sample 3 was obtained after purified through column chromatography, 0.5ml of Ni-NTA resin/buffer. Sample 4, 5, and 6 were collected after the addition of 5ml of wash buffer, 5ml of elution buffer, and the remaining elution buffer. Nanodrop spectrophotometer was used to measure the absorbance of the elution 1 at 280nm and maximal wavelength, 574nm. The samples 1 to 6 were placed in SDS-PAGE gel for gel electrophoresis process. The gel electrophoresis was done for 20minues at 200V and MW standard used was Pageruler by Germentas on figure 12. The stained protein was placed in orbital shaker for 1.5 hours and retrieved and dried at 75 ̊C for 1.5 hours.
Results:
Figure1: Fun plate for the bacterial colony
Figure2: Experimental plate with pGEM-gbr22 DNA
Figure3: Control plate with no DNA
Figure4: The bacterial colony with purple culture of BL31(DE3) and pGEM-gbr22.
Figure5: The cell pellet grown from large culture color in purple. The wet pellet weight was 0.18g.
Figure6: Extracted solutions of Elution1 and Elution2 contain purified pGEM-gbr22
Figure7: Trial 1 of protein A280 using Nanodrop spectrophotometer. Absorption at 0.318 at wavelength of 280nm.
Figure8: Trial 2 of protein A280 using Nanodrop spectrophotometer. Absorption at 0.360 at wavelength of 280nm.
Figure9: Trial 1 on UV/VIS mode of 574 with Elution1 using Nanodrop spectrophotometer. Absorption at 0.033 at wavelength of 574nm.
Figure10: Trial 2 on UV/VIS mode of 574 with Elution1 using Nanodrop spectrophotometer. Absorption at 0.032 at wavelength of 574nm.
Beer's Law Calculation (A=Ebc)
- Molecular weight of gbr22 = 25794.2 g/mol
- The average of protein measured at 280nm >> c = A/Eb = 0.339/118300 = 0.0779M (mol/L)
- The average of protein measured at 574nm >> 0.325 absorbance >> c = 0.216M (mol/L)
Yields from Nanodrop spectrophotometry
- The yield for 280nm = 0.0739 x 5= 0.3695 mg/ml
- The yield for the maximal wavelength (574nm) = 1.08mg/ml
Figure11: Gel electrophoresis using mini-PROTEAN before drying gel
Figure12: Cracked gel electrophoresis after drying gel for 1.5 hours. MW, 1,2,3,4,5,and 6 were used and 4,5,6 for the rest from partner.
Figure13: Ladder of Fermentas Pageruler MW standard (kDa)
Discussion:
The lysozyme was used to break down the cell wall of the E. Coli bacteria that pGEM-gbr22 would separated and purify the only gbr22 protein. Benzonase was used to increase fluidity or decrease viscosity of the solution of DNA or RNA. Sample 1 contained cell lysate, Sample 2 contained bacterial DNA after centrifuge, and sample 3 contained after “flow through” of the Ni-NTA solution which came from the waste of the first column run. Sample 4 contained protein and wash pellet soultion. Sample 5 and 6 contained protein and after addition of elution buffer 1 and 2. The wash buffer was made of small amount of imidazole used to ‘wash’ the pellet after the waster was removed, while elution buffer was consisted of PBX and imidazole solution. HIS tag system was often used for protein purification of E. Coli that bind to the Ni-NTA added to the solution containing protein. The protein was unbind from Ni-NTA using imidazole from elution buffer therefore it was collected into elution1 and 2. Bacterial cells were collected from centrifugation and the resulting cell pellet is lysed. The protein was purified very well after the last lab and gel image was relatively similar. Numerous errors occurred; Decanting the liquid part resulted in loss of the pellet and greater amount of buffer or imidazole solution was possibly added to the solution. Gel was cracked after gel drying because cell paper was not strong enough during the process
Conclusions:
pGEM-gbr22 protein using recombinant DNA and the bacterial E. Coli was used during entire lab and purified. The molecular weight of the purified protein was estimated to be around 27kDa. The percent of purity was around 75 to 80%. The protein was left for further protein lab. Molecular weight and absorbency using gel electrophoresis and spectrophotometer of protein can be figured in the future and enzyme assays could be created to explore the function of protein and find out the inhibitory molecules.
References:
[1] Lesley, S.; High-Throughput Proteomics: ProteinExpression and Purification in the Postgenomic World. Genomics Institute. 2001, 22(2): 159-164.
[2] Tan, Song.; A Modular Polycistronic Expression System for Overexpressing Protein Complexes in Escherichia coli.Center for Gene Regulation. 2001, 21(1): 224-234. NEB, beakers, several test tubes, petri dishes, centrifuge tubes, pipettors and tips and, Erlenmeyer flasks, spectrophotometer (Thermo Scientific, Wilmington, DE), gel electrophoresis device (mini-PROTEAN), centrifuge machine, a and other lab equipments. Procedure was divided into three parts: bacterial protein expression, purification, and characterization. E. Coli bacteria was prepared and 25ul of bacteria was added into 2 transformation tubes: one control and other with DNA. Petri dishes were used and 200ul of SOC media were added after few ice and heat shock process. 50ul of mixture were added to 2 petri dishes and labeled: one control and other with DNA. Plates were stored in 37 ̊C incubator overnight. Bacterial colonies were added to LB broth and left in incubator for 7 hours. Ampicillin was added to the culture in Erlenmeyer flask and left in incubator for a day to grow. 500ul of purple sample 1 was collected from the culture and centrifuged for 5 minutes at 5000 rpm at 4 ̊C. 2.5ml of 1x PBS solution was added to the tube and lysozyme was also added to make the final concentration of 1mg/ml. Wet pellet was weighted 12 0.18g after centrifuge. Sample2 was obtained after the centrifuge of 2ul of benzonase added to solution. The remained liquid was syringe filtered. Wash and elution buffers were made from 1 PBS and 20mM imidazole and 1x PBS and 250mM imidazole. Sample 3 was obtained after purified through column chromatography, 0.5ml of Ni-NTA resin/buffer. Sample 4, 5, and 6 were collected after the addition of 5ml of wash buffer, 5ml of elution buffer, and the remaining elution buffer. Nanodrop spectrophotometer was used to measure the absorbance of the elution 1 at 280nm and maximal wavelength, 574nm. The samples 1 to 6 were placed in SDS-PAGE gel for gel electrophoresis process. The gel electrophoresis was done for 20minues at 200V and MW standard used was Pageruler by Germentas on figure 12. The stained protein was placed in orbital shaker for 1.5 hours and retrieved and dried at 75 ̊C for 1.5 hours. e use of recombinant proteins in research has increased as the development of new technologies and analysis of protein structure and function used for amplification and purification. Protein expression and purification are key processes in study of genomic information, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the high-throughput screening technologies commonly used in pharmaceutical development [1]. E.Coli bacterial protein would overexpressed and purified for affinity, ion-exchange crystallography, enzyme inhibition assays and other techniques [2]. Concentration was calculated using spectrophotometer and Beer’s law. Three protein labs were to overexpress, purigy, and characterize the recombinant pGEM-gbr22 protein.
Title: GBR22 Protein experiments of expression, purification, and characterization.
Introduction:
The use of recombinant proteins in research has increased as the development of new technologies and analysis of protein structure and function used for amplification and purification. Protein expression and purification are key processes in study of genomic information, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the high-throughput screening technologies commonly used in pharmaceutical development [1]. E.Coli bacterial protein would overexpressed and purified for affinity, ion-exchange crystallography, enzyme inhibition assays and other techniques [2]. Concentration was calculated using spectrophotometer and Beer’s law. Three protein labs were to overexpress, purify, and characterize the recombinant pGEM-gbr22 protein.
Materials & Methods:
As safety precautions, goggles, gloves, and lab coats were worn even before the pre-lab calculation and food and drinks were prohibited in lab area. The materials used were bacteria E. Coli BL21 (DE3) strain, pGEM-gbr22 protein from NEB, beakers, several test tubes, petri dishes, centrifuge tubes, pipettors and tips and, Erlenmeyer flasks, spectrophotometer (Thermo Scientific, Wilmington, DE), gel electrophoresis device (mini-PROTEAN), centrifuge machine, a and other lab equipments. Procedure was divided into three parts: bacterial protein expression, purification, and characterization. E. Coli bacteria was prepared and 25ul of bacteria was added into 2 transformation tubes: one control and other with DNA. Petri dishes were used and 200ul of SOC media were added after few ice and heat shock process. 50ul of mixture were added to 2 petri dishes and labeled: one control and other with DNA. Plates were stored in 37 ̊C incubator overnight. Bacterial colonies were added to LB broth and left in incubator for 7 hours. Ampicillin was added to the culture in Erlenmeyer flask and left in incubator for a day to grow. 500ul of purple sample 1 was collected from the culture and centrifuged for 5 minutes at 5000 rpm at 4 ̊C. 2.5ml of 1x PBS solution was added to the tube and lysozyme was also added to make the final concentration of 1mg/ml. Wet pellet was weighted 12 0.18g after centrifuge. Sample2 was obtained after the centrifuge of 2ul of benzonase added to solution. The remained liquid was syringe filtered. Wash and elution buffers were made from 1 PBS and 20mM imidazole and 1x PBS and 250mM imidazole. Sample 3 was obtained after purified through column chromatography, 0.5ml of Ni-NTA resin/buffer. Sample 4, 5, and 6 were collected after the addition of 5ml of wash buffer, 5ml of elution buffer, and the remaining elution buffer. Nanodrop spectrophotometer was used to measure the absorbance of the elution 1 at 280nm and maximal wavelength, 574nm. The samples 1 to 6 were placed in SDS-PAGE gel for gel electrophoresis process. The gel electrophoresis was done for 20minues at 200V and MW standard used was Pageruler by Germentas on figure 12. The stained protein was placed in orbital shaker for 1.5 hours and retrieved and dried at 75 ̊C for 1.5 hours.
Results:
Beer's Law Calculation (A=Ebc)
- Molecular weight of gbr22 = 25794.2 g/mol
- The average of protein measured at 280nm >> c = A/Eb = 0.339/118300 = 0.0779M (mol/L)
- The average of protein measured at 574nm >> 0.325 absorbance >> c = 0.216M (mol/L)
Yields from Nanodrop spectrophotometry
- The yield for 280nm = 0.0739 x 5= 0.3695 mg/ml
- The yield for the maximal wavelength (574nm) = 1.08mg/ml
Discussion:
The lysozyme was used to break down the cell wall of the E. Coli bacteria that pGEM-gbr22 would separated and purify the only gbr22 protein. Benzonase was used to increase fluidity or decrease viscosity of the solution of DNA or RNA. Sample 1 contained cell lysate, Sample 2 contained bacterial DNA after centrifuge, and sample 3 contained after “flow through” of the Ni-NTA solution which came from the waste of the first column run. Sample 4 contained protein and wash pellet soultion. Sample 5 and 6 contained protein and after addition of elution buffer 1 and 2. The wash buffer was made of small amount of imidazole used to ‘wash’ the pellet after the waster was removed, while elution buffer was consisted of PBX and imidazole solution. HIS tag system was often used for protein purification of E. Coli that bind to the Ni-NTA added to the solution containing protein. The protein was unbind from Ni-NTA using imidazole from elution buffer therefore it was collected into elution1 and 2. Bacterial cells were collected from centrifugation and the resulting cell pellet is lysed. The protein was purified very well after the last lab and gel image was relatively similar. Numerous errors occurred; Decanting the liquid part resulted in loss of the pellet and greater amount of buffer or imidazole solution was possibly added to the solution. Gel was cracked after gel drying because cell paper was not strong enough during the process
Conclusions:
pGEM-gbr22 protein using recombinant DNA and the bacterial E. Coli was used during entire lab and purified. The molecular weight of the purified protein was estimated to be around 27kDa. The percent of purity was around 75 to 80%. The protein was left for further protein lab. Molecular weight and absorbency using gel electrophoresis and spectrophotometer of protein can be figured in the future and enzyme assays could be created to explore the function of protein and find out the inhibitory molecules.
References:
[1] Lesley, S.; High-Throughput Proteomics: ProteinExpression and Purification in the Postgenomic World. Genomics Institute. 2001, 22(2): 159-164.
[2] Tan, Song.; A Modular Polycistronic Expression System for Overexpressing Protein Complexes in Escherichia coli. Center for Gene Regulation. 2001, 21(1): 224-234. NEB, beakers, several test tubes, petri dishes, centrifuge tubes, pipettors and tips and, Erlenmeyer flasks, spectrophotometer (Thermo Scientific, Wilmington, DE), gel electrophoresis device (mini-PROTEAN), centrifuge machine, a and other lab equipments. Procedure was divided into three parts: bacterial protein expression, purification, and characterization. E. Coli bacteria was prepared and 25ul of bacteria was added into 2 transformation tubes: one control and other with DNA. Petri dishes were used and 200ul of SOC media were added after few ice and heat shock process. 50ul of mixture were added to 2 petri dishes and labeled: one control and other with DNA. Plates were stored in 37 ̊C incubator overnight. Bacterial colonies were added to LB broth and left in incubator for 7 hours. Ampicillin was added to the culture in Erlenmeyer flask and left in incubator for a day to grow. 500ul of purple sample 1 was collected from the culture and centrifuged for 5 minutes at 5000 rpm at 4 ̊C. 2.5ml of 1x PBS solution was added to the tube and lysozyme was also added to make the final concentration of 1mg/ml. Wet pellet was weighted 12 0.18g after centrifuge. Sample2 was obtained after the centrifuge of 2ul of benzonase added to solution. The remained liquid was syringe filtered. Wash and elution buffers were made from 1 PBS and 20mM imidazole and 1x PBS and 250mM imidazole. Sample 3 was obtained after purified through column chromatography, 0.5ml of Ni-NTA resin/buffer. Sample 4, 5, and 6 were collected after the addition of 5ml of wash buffer, 5ml of elution buffer, and the remaining elution buffer. Nanodrop spectrophotometer was used to measure the absorbance of the elution 1 at 280nm and maximal wavelength, 574nm. The samples 1 to 6 were placed in SDS-PAGE gel for gel electrophoresis process. The gel electrophoresis was done for 20minues at 200V and MW standard used was Pageruler by Germentas on figure 12. The stained protein was placed in orbital shaker for 1.5 hours and retrieved and dried at 75 ̊C for 1.5 hours.
e use of recombinant proteins in research has increased as the development of new technologies and analysis of protein structure and function used for amplification and purification. Protein expression and purification are key processes in study of genomic information, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the high-throughput screening technologies commonly used in pharmaceutical development [1]. E.Coli bacterial protein would overexpressed and purified for affinity, ion-exchange crystallography, enzyme inhibition assays and other techniques [2]. Concentration was calculated using spectrophotometer and Beer’s law. Three protein labs were to overexpress, purigy, and characterize the recombinant pGEM-gbr22 protein.